J. Phys. Chem. A 2003, 107, 10787-10797
10787
Picosecond Time-Resolved Coherent Anti-Stokes Raman Spectroscopy of the Artificial Bacteriorhodopsin Pigment, BR6.11† Andrew C. Terentis, Yidong Zhou, and George H. Atkinson* Department of Chemistry and Optical Sciences Center, UniVersity of Arizona, Tucson, Arizona 85721
Laszlo Ujj Department of Physics, UniVersity of West Florida, Pensacola, Florida 32514 ReceiVed: May 13, 2003; In Final Form: July 24, 2003
The picosecond molecular dynamics in an artificial bacteriorhodopsin (BR) pigment, BR6.11, are measured by picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS) and picosecond transient absorption (PTA). The BR6.11 pigment contains a structurally modified retinal chromophore with a sixmembered carbon ring bridging the C11dC12-C13 bonds, which both locks the C11dC12 bond in the trans configuration and prevents rotation about the C12-C13 bond. The changes in the vibrational degrees of freedom of the retinal attributable to the six-membered carbon ring are found in the picosecond resonance CARS (PR/CARS) spectrum of ground-state BR6.11. Normal mode assignments for more than forty BR6.11 vibrational features are made through comparisons with the PR/CARS data from native BR-570 (previously analyzed in terms of the selective isotopic substitution of the retinal). PTR/CARS spectra from two intermediates (J6.11 and K6.11), observed by PTA to appear during the initial 200 ps of the BR6.11 photocycle, reveal distinct retinal structures for each. The retinal in J6.11 contains delocalized (i.e., vibrational degrees of freedom not well described by normal modes and spanning major regions of the retinal), out-of-plane motion, and a highly twisted all-trans polyene configuration while K6.11 contains a retinal in which vibrational degrees of freedom have relaxed into well-defined, out-of-plane normal modes and a less twisted (though markedly nonplanar), 13-cis configuration. These vibrational data show that C13dC14 isomerization is not the first structural change to occur in the BR6.11 photocycle, but rather is the principal structural change during the J6.11 to K6.11 transformation. The similarity of the retinal structural dynamics in the BR6.11 and native BR-570 photocycles demonstrates that subpicosecond, torsional motion within the polyene precedes C13d C14 isomerization and is critical for initiating the BR photoreactivity underlying their biochemical function. The structural dynamics comprising the initial 200 ps of the BR6.11 photocycle, as derived from these PTR/ CARS data, are described well by a three-state model in which J6.11 and K6.11 are assigned as excited and ground electronic states, respectively.
Introduction The purple membrane of the archaebacterium, Halobacterium salinarum, contains a single trans-membrane protein, bacteriorhodopsin (BR), that stores and utilizes absorbed light energy to transport protons across the cell membrane.1-3 The resulting potential gradient created across the cell membrane drives ATP synthesis.3 The high efficiency of the BR molecular mechanism, as well as its role as a prototype for other, more complicated ion transporting proteins, underlies much of the biochemical interest in BR. Although many features of the energy storage mechanism within BR are reasonably well characterized,4,5 the dynamics of the initial structural changes in the retinal chromophore are still to be fully understood. Of particular interest are those structural changes occurring during the initial femto/picosecond period of the BR photocycle. Trans to cis isomerization of the C13dC14 bond has been widely viewed in earlier models of the BR photocycle as the primary molecular event.1,6 These models * To whom correspondence should be addressed. E-mail: atkinsog@ u.arizona.edu. † Part of the special issue “Charles S. Parmenter Festschrift”.
also assume that all-trans to 13-cis isomerization within retinal occurs prior to the 0.5 ps appearance of the J-625 intermediate.1,6 Recently,7 however, picosecond time-resolved coherent antiStokes Raman spectra (PTR/CARS) indicate that J-625 contains an all-trans-like retinal. Specifically, the C-C stretching band (“fingerprint”) pattern, used widely in vibratonal spectroscopy to identify the retinal configuration,1 remains almost unchanged as J-625 is formed from BR-570 (all-trans), but changes significantly as K-590 (13-cis) subsequently (3.5 ps) appears.7 Thus, J-625 is an intermediate in which the retinal structure has undergone significant changes relative to BR-570 (vide infra), but C13dC14 isomerization is not one of them. The change from all-trans to 13-cis retinal occurs during the J-625 to K-590 transformation. The structural properties of the J-625 retinal that distinguish it from those in BR-570 and K-590 include the following: (i) increased electron density in the CdC bonds (increased CdC stretching mode frequencies), (ii) significant “delocalized” hydrogen out-of-plane (HOOP) motion, and (iii) a Schiff-base bonding environment similar to that of BR-570, but distinctively different from that in K-590. These vibrational CARS features indicate that J-625 has a highly distorted (twisted) retinal
10.1021/jp030612g CCC: $25.00 © 2003 American Chemical Society Published on Web 09/20/2003
10788 J. Phys. Chem. A, Vol. 107, No. 49, 2003
Figure 1. (A) Chemical structures of retinal chromophores in BR570, BR5.12, BR6.9, and BR6.11 and (B) absorption spectra of BR570 (solid line, intensity maximum at 568 nm), BR5.12 (dotted line, intensity maximum at 578 nm), BR6.9 (dot-dash line, intensity maximum at 572 nm), and BR6.11 (dashed line, intensity maximum at 556 nm). The pump (ω1 ) 663 nm) and probe (ωs ) 700-750 nm) wavelengths used to obtain PR/CARS spectra from BR6.11 are shown at the bottom together with the spectral region (595-635 nm) where the ωas signal appears.
conformation, an observation that is consistent with earlier timeresolved resonance Raman results.8 The third type of difference also supports the idea that trans to cis retinal isomerization does not occur until K-590 is formed, whereby the retinal Schiff base proton moves to a different position within the protein pocket. “Delocalized” out-of-plane retinal motion connotes vibrational energy not well described by normal modes. Appearing as highly broadened (>50 cm-1) vibrational features, delocalized out-ofplane motion is distributed over major regions of the retinal, and therefore differs from out-of-plane motion observed as unbroadened (50 ns, respectively.20 Independently, BR6.9 has been shown to have biochemical activity similar to that in BR-570.15 Other than slowing the formation and decay rates for the intermediates, these data show that restricting retinal motion at the C9dC10-C11 bonds does not significantly alter the photocycle mechanism of BR. The higher (temporal and spectral) resolution of the PR/CARS and PTR/CARS data from BR6.11 presented here (as compared to previous time-resolved RR data18) are used to examine the role of C11dC12-C13 bond torsions and C13dC14 isomerization. Distinct retinal structures are identified from the spectra of BR6.11, J6.11, and K6.11. Specifically, C13dC14 isomerization occurs in the transition from J6.11 to K6.11 while J6.11 is characterized by the same intense, delocalized HOOP motions that are observed in J-625. These experimental CARS results support the conclusions reached from earlier CARS studies that strong, delocalized, out-of-plane vibrations of the polyene chain precede the trans-cis isomerization of the C13dC14 bond.
Artificial Bacteriorhodopsin Pigment: BR6.11
J. Phys. Chem. A, Vol. 107, No. 49, 2003 10789
Finally, these CARS results are discussed in terms of a threestate model used to describe the dynamics controlling the initial stages of the BR photoreactivity in both BR6.11 and in BR pigments in general. The consistency between the PTR/CARS data presented here and a three-state model indicates that J6.11, and J species in general, be assigned as excited electronic states, while K6.11, and K species in general, be assigned as ground electronic states. Materials The BR samples are grown from a cell line of Halobacterium salinarum and are purified according to established methods.22,24 The purple membrane typically has an absorbance ratio of 1.5 (protein absorbance (280 nm)/retinal absorbance (570 nm)) and is used without further purification. The isolated bacterio-opsin is obtained by removing the all-trans retinal chromophore from BR-570 by a bleaching procedure: BR-570 is bleached for ∼48 h by focusing the output of a 500-W projector lamp through a 450-nm cutoff filter onto a light-adapted BR solution containing 1 M NH3OHCl, 1 M NaOH and 1 M NaCl buffered at pH ) 7.0. After bleaching, the sample is centrifuged and washed with 10 mM K2HPO4/dithiothreitol (DTT) buffer and 5% v/v solution of bovine serum albumin (BSA) to eliminate the hydroxylamine and any residual all-trans retinal. BR6.11 is prepared by reconstituting the remaining opsin with the structurally modified retinal E-11,20-ethanoretinal (Ret6.11). The Ret6.11 is dissolved in ethanol and added to the buffered opsin solution (
